Methods of isolating bipotent hepatic progenitor cells

ABSTRACT

A method of obtaining a mixture of cells enriched in hepatic progenitors is developed which comprises methods yielding suspensions of a mixture of cell types, and selecting those cells that are classical MHC class I antigen(s) negative and ICAM-1 antigen positive. The weak or dull expression of nonclassical MHC class I antigen(s) can be used for further enrichment of hepatic progenitors. Furthermore, the progenitors can be selected to have a level of side scatter, a measure of granularity or cytoplasmic droplets, that is higher than that in non-parenchymal cells, such as hemopoietic cells, and lower than that in mature parenchymal cells, such as hepatocytes. Furthermore, the progeny of the isolated progenitors can express alpha-fetoprotein and/or albumin and/or CK19. The hepatic progenitors, so isolated, can grow clonally, that is an entire population of progeny can be derived from one cell. The clones of progenitors have a growth pattern in culture of piled-up aggregates or clusters. These methods of isolating the hepatic progenitors are applicable to any vertebrates including human. The hepatic progenitor cell population is expected to be useful for cell therapies, for bioartificial livers, for gene therapies, for vaccine development, and for myriad toxicological, pharmacological, and pharmaceutical programs and investigations.

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation of Ser. No. 10/139,231 filed May 7,2002, which is a divisional of U.S. application Ser. No. 09/678,953filed Oct. 3, 2000, which claims priority to 60/157,052 filed Oct. 1,1999, the disclosures of which are incorporated by reference herein intheir entireties.

FIELD OF THE INVENTION

The present invention relates to novel cell surface markers thatdistinguish hepatic cells from hematopoietic cells. In particular, theinvention relates to methods of isolatingbipotent hepatic progenitorcells with a unique phenotype that includes cells that are negative forclassical major histocompatibility complex (MHC) class I antigen,positive for the intercellular adhesion molecule 1 (ICAM-1), and dullpositive for nonclassical MHC class I antigen(s). Moreover, theinvention relates to the hepatic progenitor and hepatic stem cellsproduced by the methods of the invention.

DESCRIPTION OF RELATED ART

Identification of multipotential progenitor cell populations inmammalian tissues is important both for clinical and commercialinterests and also for understanding of developmental processes andtissue homeostasis. Progenitor cell populations are ideal targets forgene therapy, cell transplantation and for tissue engineering ofbioartificial organs (Millar, A. D. 1992 Nature 357, 455; Langer, R. andVacanti, J. P. 1993 Science 260,920; Gage, F. H. 1998 Nature 392, 18).

The existence of tissue-specific, “determined” stem cells or progenitorshaving high growth potential and/or pluripotentiality is readilyapparent from studies on hematopoietic stem cells (Spangrude et al. 1988Science 241, 58), neuronal stem cells (Davis, A. A., and Temple, S. 1994Nature 372, 263; Stemple, D. L., and Anderson, D. J. 1992 Cell 71, 973)and epidermal stem cells (Jones, P. H., and Watt, F. M. 1993 Cell73,713), each having been identified clonally by using the particularmethods appropriate for that tissue. These progenitors are regarded asthe cells responsible for normal hematopoietic, neuronal or epidermaltissue homeostasis and for regenerative responses 25 after severe injury(Hall, P. A., and Watt, F. M. 1989 Development 106, 619).

The mammalian adult liver has a tremendous capacity to recover aftereither extensive hepatotoxic injury or partial hepatectomy (Fishback, F.C. 1929 Arch. Pathol. 7, 955); (Higgins, G. M., and Anderson, R. M. 1931Arch. Pathol. 12, 186), even though the liver is usually a quiescenttissue without rapid turnover. Data from recent studies in the mousehave been interpreted to suggest that adult parenchymal cells have analmost unlimited growth potentiality as assayed by serialtransplantation experiments (Overturf et al. 1997 Am. J. Pathol. 151,1273); (Rhim, J. A. et al. 1994 Science 263, 1149). These experimentsmade use of heterogeneous liver cell population limiting the ability toprove that the growth potential observed derived from adult parenchymalcells, from a subpopulation of adult parenchymal cells and/or fromnon-parenchymal cells (i.e. progenitors). Furthermore, the studies showno evidence for biliary epithelial differentiation, since the hosts usedhad either albumin-urokinase transgenes or, in the other case, atyrosine catabolic enzyme deficiency; both types of hosts haveconditions that selected for the hepatocytic lineage. Therefore, theassay was incapable of testing for bipotent cell populations.

Several histological studies establish that early hepatic cells frommidgestational fetuses have a developmental bipotentiality todifferentiate to bile duct epithelium as well as to mature hepatocytes(Shiojiri, N. 1997 Microscopy Res. Tech. 39, 328-35). Hepaticdevelopment begins in the ventral foregut endoderm immediately after theendodermal epithelium interacts with the cardiogenic mesoderm (Douarin,N. M. 1975 Medical Biol. 53, 427); (Houssaint, E. 1980 Cell Differ. 9,269). This hepatic commitment occurs at embryonic day (E) 8 in themouse. The initial phase of hepatic development becomes evident with theinduction of serum albumin and alpha-fetoprotein mRNAs in the endodermand prior to morphological changes (Gualdi, R. et al. 1996 Genes Dev.10, 1670). At E 9.5 of mouse gestation, the specified cells thenproliferate and penetrate into the mesenchyme of the septum transversumwith a cord-like fashion, forming the liver anlage. Although the livermass then increases dramatically, the increase in mass is due largely tohematopoietic cells, which colonize the fetal liver at E10 in the mouse(Houssaint, E. 1981 Cell Differ. 10, 243) and influence the hepaticcells to show an extremely distorted and irregular shape (Luzzatto, A.C. 1981 Cell Tissue Res. 215, 133). Interestingly, recent data fromgene-targeting mutant mice indicates that impairment of a number ofgenes has led to lethal hepatic failure, apoptosis and/or necrosis ofparenchymal cells between E12 to E15 (Gunes, C. et al. 1998 EMBO J. 17,2846; Hilberg, F. et al. 1993 Nature 365, 1791; Motoyama, J. et al. 1997Mech. Dev. 66, 27; Schmidt, C. et al. 1995 Nature 373, 699). Especiallygene disruptions that are part of the stress-activated cascade(Ganiatsas, S. et al. 1998. Proc. Natl. Acad. Sci USA 95, 6881; Nishina,H. et al. 1999 Development 126, 505) or anti-apoptotic cascade (Beg, A.et al. 1995 Nature 376, 167; Li, Q. et al. 1999 Science 284, 321;Tanaka, M. et al. 1999. Immunity 10, 421) can result in severelyimpaired hepatogenesis, not hematopoiesis, in spite of the broadexpression of the inactivated gene. It is not clear whether hepaticcells are intrinsically sensitive to developmental stress stimuli orthat the particular microenvironment in fetal liver per se causes suchdestructive effects (Doi, T. S. et al 1999 Proc. Natl. Acad. Sci. USA96, 2994). On the other hand, the basic architecture of adult liver isdependent on the appearance of the initial cylinder of bile ductepithelium surrounding the portal vein (Shiojiri, N. 1997 MicroscopyRes. Tech. 39, 328). Immunohistologically, the first sign of thedifferentiation of intrahepatic bile duct epithelial cells is theexpression of biliary-specific cytokeratin (CK). CK proteins, thecytoplasmic intermediate filament (IF) proteins of epithelial cells, areencoded by a multigene family and expressed in a tissue- anddifferentiation-specific manner (Moll, R. et al. 1982 Cell 31, 11). CK19is one of the most remarkable biliary markers, because adult hepatocytesdo not express CK19 at all, whereas adult biliary epithelial cells doexpress this protein. Only CK8 and CK18 are expressed through earlyhepatic cells to adult hepatocytes (Moll, R. et al. 1982 Cell 31, 11).At E15.5 in the rat development, corresponding to E14 in the mouse, thebiliary precursors are heavily stained by both CK18 and CK8 antibodies,and some biliary precursors express CKl9. As development progresses,maturing bile ducts gradually express CK7 in addition to CK19 and losethe expression of albumin (Shiojiri, N. et al. 1991 Cancer Res. 51,2611). Although hepatic cells as early as E13 in the rat are thought tobe a homogeneous population, it remains to be seen whether all earlyhepatic cells can differentiate to biliary epithelial cell lineage, andhow their fates are determined. Definitive lineage-marking studies, suchas those using retroviral vectors, have not been done for hepatic cells,and clonal culture conditions requisite for the demonstration of anybipotent hepatic progenitor cells have not been identified.

For clonal growth analyses, one major obstacle is the explosiveexpansion of hematopoietic cells, marring the ability to observe ex vivoexpansion of hepatic cells. Therefore an enrichment process for thehepatic population must be used. Although the surface markers to be ableto fractionate the hematopoietic cells in fetal liver have beeninvestigated in detail (Dzierzak, E. et al. 1998 Immunol. Today 19,228-36), those for hepatic progenitor cells are still poorly defined,since the studies are still in their infancy (Sigal, S. et al. 1994Hepatology 19, 999). Furthermore, the ex vivo proliferation conditionstypically used for adult liver cells result in their dedifferentiationwith loss of tissue-specific functions such as albumin expression(Block, G. D. et al. 1996 J. Cell Biol. 132, 1133). A somewhat improvedability to synthesize tissue-specific mRNAs and ability to regulatetissue-specific genes fully post-transcriptionally occurs only in livercells maintained in the absence of serum and with a defined mixture ofhormones, growth factors and/or with certain extracellular matrixcomponents (Jefferson, D. M. et al. 1984. Mol. Cell. Biol. 4, 1929; EnatR, et al 1984, 81, 1411). Proliferating fetal hepatic cells, however,maintain the expression of such serum proteins in vivo.

In addition to hepatic progenitor cells, the fetal liver in many speciescontains hematopoietic progenitor cells. The hematopoictic progenitorcells and hematopoietic cells express major histocompatability (MHC)antigens on their surfaces. The nomenclature of MHC has not beenentirely standardized. Thus the classical MHC class I antigen may alsobe designated MHC class Ia or MHC class IA. Similarly, the non-classicalMHC class I antigen may also be designated MHC class Lb or MHC class LB.

Among work on MHC antigens, U.S. Pat. No. 5,679,340 to Chappel claimsmodification of cell surface antigens including MHC by bindingantibodies to two antigenic epitopes. In contrast, Chappel fails toteach that MHC and other antigens can be used for isolation ofprogenitor cells.

Others have attempted to grow hepatocytes in vitro. U.S. Pat. No.5,510,254 to Naughton et al. claims the culture of hepatocytes dependson a three-dimensional framework of biocompatible but non-livingmaterial. Thus there is an unmet need for culture conditions with noartificial framework and providing the condition for hepatic progenitorsto be expanded and cultured. Furthermore, there is an unmet need formethods of cloning of hepatic progenitors with biopotentialdifferentiation capability, where the cells would be suitable for use ascomponents of a bio-artificial liver, for testing of hepatotoxins anddrug development, among other uses.

U.S. Pat. No. 5,559,022 to Naughton et al., claims liver reserve cellsthat bind Eosin Y, a stain that was used to characterize the “reservecells.” U.S. Pat. No. 5,559,022 does not use well-established markersfor identification of liver reserve cells, nor provide methods forclonal expansion of reserve cells, nor provided markers by which toisolate viable liver reserve cells. Thus, there is an unfilled need formethods to isolate and culture cells that have many features essentialto hepatic progenitors, including expression of specific markers and thepotential to differentiate into either hepatocytes or biliary cells.Equally needed are methods for clonal growth of the hepatic progenitors.Clonal growth is essential as a clear and rigorous distinction andidentification of pluripotent hepatic progenitors.

The present inventors have recognized the inadequacy of growing matureliver cells, such as hepatocytes, rather than the far more usefulhepatic progenitors. They have carefully defined the isolationparameters for hepatic progenitors and requirements for clonal growth.The progenitor cells and the methods for selecting and culturing theprogenitors have many uses, including utility in medicine for treatmentof patients with liver failure, and utility for evaluation of toxicityagents, and utility for evaluation of drugs.

SUMMARY OF THE INVENTION

The present invention relates to a method of isolating hepatic bipotentprogenitor cells where the cells do not express the classical MHC classI antigen (MHC class Ia antigen) and do express the ICAM antigen orICAM-1 antigen. Furthermore, the hepatic bipotent progenitor canoptionally express nonclassical MHC class I antigen(s) (MHC class Ibantigen) containing monomorphic epitope of MHC class I. Progenitors fromseveral tissues can be used, including, but not limited to, liver. Thus,the invention relates to a method of isolating hepatic progenitor cellsthat are classical MHC class I negative and, optionally, ICAM-1positive. Likewise, the present invention relates to a method ofisolating progenitor cells, where the cells express the phenotype ofICAM-1 positive but classical MHC class I negative, by removing cellsthat express the phenotype classical MHC class I positive. The dullexpression of nonclassical MHC class I can be used for further isolationof progenitor cells. Preferably, the invention relates to a method ofisolating and cloning hepatic pluripotent progenitor cells. The hepaticpluripotent progenitor cells may be of any vertebrate species includingfish, amphibian, reptilian, avian, and mammalian, and more preferablymammalian. Even more preferably, the hepatic pluripotent progenitorcells are primate, pig, rat, rabbit, dog, or mouse in origin. Mostpreferably the pluripotent progenitor cells are human in origin. Thevery most preferable method yields hepatic progenitors that are bipotenthepatic progenitors. Thus the bipotent hepatic progenitors candifferentiate, or their progeny can differentiate, into eitherhepatocytes or biliary cells.

A cell population enriched in progenitors can be obtained by a method offirst obtaining a cell suspension of vertebrate cells. Then,sequentially in either order, or substantially simultaneously, the cellsthat express at least one MHC class Ia antigen and those that express anICAM antigen, are removed from the cell suspension to provide a mixtureof cells enriched in progenitors. Equally, a mixture of vertebrateembryonic stem cell can be obtained that is enriched in hepaticprogenitors by providing a vertebrate embryonic stem cell, expanding theembryonic stem cell to give embryonic stem cell progeny and isolatingthose embryonic stem cell progeny which express ICAM antigen and do notexpress MHC class Ia antigen.

All methods of separation by physical, immunological, and cell culturemeans known in the art are included in the invention. The methods ofseparation specifically include the immunoseparations. Immunoseparationscan be flow cytometry after interaction with a labeled antibody.Immunoseparation methods also include affinity methods with antibodiesbound to magnetic beads, biodegradable beads, non-biodegradable beads,to panning surfaces including dishes, and to combinations of thesemethods.

Furthermore, the hepatic progenitor and bipotent stem cells, and theirprogeny, can optionally express other phenotypes, including, but in noway limited to alpha-fetoprotein, albumin, a higher side scatter thanhematopoietic cells from fetal liver, or a pattern of growth as cellsthat pile up.

Hepatic stem cells are cells that might or might not expressalpha-fetoprotein or albumin but give rise to cells that expressalpha-fetoprotein and albumin or biliary markers such as CK19.

The invention also relates to a method for the identification ofprogenitor cells, preferably hepatic progenitor cells, by exposing livercells to a means of detecting a MHC class I phenotype in combinationwith ICAM-1 expression, and identifying those cells within thepopulation that do not express classical MHC class I antigen. Likewise,other markers of progenitor or hepatic phenotypes such asalpha-fetoprotein can be detected.

The invention additionally relates to hepatic stem and progenitor cells,and their progeny, characterized by a phenotype of classical MHC class Inegative and ICAM-1 positive, which cells can optionally express otherphenotypes, including, but in no way limited to nonclassical MHC class Idull positive, a higher side scatter than hematopoietic cellsprogenitors, or a pattern of growth as cells that pile up. The progenycan express alpha-fetoprotein, albumin, or CK 19. The progeny of thehepatic stem and progenitor cells so isolated can retain the parentalphenotype and optionally can develop and express additional phenotypes.In particular, the progeny cells can optionally express the hepatocytephenotype and the biliary cell phenotype. Among other features, thehepatocyte phenotype is characterized by expression of albumin. Amongother features, the biliary cell phenotype is characterized byexpression of CK 19.

The composition of hepatic progenitors, their progeny, or a combinationof the progenitors and their progeny can also comprise cells that weaklyexpress at least on MHC class lb antigen, exhibit a higher side scatterin flow cytometry than non-parenchymal cells, and express a polypeptideconsisting of alpha-fetoprotein, albumin, CK 19, or combinations thereofThe composition can be derived from endoderm or bone marrow. In thiscomposition, the endoderm tissue can be liver, pancreas, lung, gut,thyroid, gonad, or combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1C is a characterization of hepatic cell lines from day 15 fetalrat liver.

FIG. 2A-2F is an assay of colony formation on feeder cells.

FIG. 3A-3X is an expression of rat cell surface antigens on varioushepatic cell lines in adult liver cells.

FIGS. 4A1-4D4 depicts phenotypic analysis of E13 fetal rat livers.

FIG. 5A-5D is characterization of hepatic colonies in the absence andpresence of EGF.

FIG. 6A-6B depicts induction of CK19 expression on RT1A1-hepatic cells.

FIG. 7 is a schematic representation of hepatic colony formation on STO5feeder cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The instant invention is a process for isolation of progenitor cells anda composition comprising progenitor cells. In one embodiment, theinvention is a process for the identification, isolation, and clonalgrowth of hepatic stem cells and of the hepatic progenitor cells. Theprocess involves exposing mixed cell populations derived from anendodermal tissue such as liver to antibodies specific for an ICAM, forexample ICAM-1, an adhesion protein, and classical MHC class I antigen,an antigen that characterizes hematopoietic cells and most othernucleated cells but that is substantially absent on the cell surface ofhepatic stem cells and progenitors proper. The cells can be from anyendodermal tissue, including but not limited to liver, pancreas, lung,gut, thyroid, gonad, or from a liver or from a whole organism. Anymethod of isolating hepatic stem and other early hepatic progenitorcells is acceptable, including by affinity-based interactions, e.g.,affinity panning, by immunosurgery in combination with complement orwith flow cytometry. The flow cytometry separation can also be based onintermediate levels of antigen expression, for example of nonclassicalMHC class I antigens. In a yet more preferred embodiment, the processinvolves, in addition, selecting for cells that show relatively highside scatter (SSC), a parameter dependent on cellular granularity oramount of cytoplasmic lipid droplets, a feature of hepatic cells. TheSSC in the hepatic progenitors is higher than in other non-parenchymalcells, such as hematopoietic cells or stromal cells in fetal liver, butlower than in mature parenchymal cells such as those in adult liver. Inaddition, other markers expressed on alpha-fetoprotein (AFP)-positiveprogenitor cells, such as CD34, CD38, CD14, and/or CD117, can be used inisolating bipotent progenitor cells. Likewise, other markers for theremoval of non-hepatic progenitor cells, including, but not limited tored blood cell antigen (such as glycophorin A on red blood cells inhuman liver), immunoglobulin F_(c) receptors, MHC class II antigens, ABOtype markers, CD2, CD3, CD4, CD7, CD8, CD9, CD11a, CD11b, CD11c, CD15,CD16, CD19, CD20, CD28, CD32, CD36, CD42, CD43, CD45, CD56, CD57, CD61,CD74, CDw75 can be used. Furthermore, other techniques known in the artmay be used as components of processes used to isolate progenitor cells,including, but not limited to: ablative techniques including laserablation, density separation, sedimentation rate separation includingzonal centrifugation, cell elutriation, selective adherence, molecularweighting including cell weighting with tetrazolium salts, size sieving,selective propagation, selective metabolic inhibition including use ofcytotoxins, and multi-factor separation.

In one preferred embodiment of the invention the progenitor cells areobtained from a fetus, a child, an adolescent, or an adult.

It is a preferred embodiment of the instant invention that hepatic cellsbe selectively grown in a seran-free, hormone-supplemented, definedmedium. It is further preferred that hepatic cells be selectively grownin culture using a layer of feeder cells, where those feeder cells arefibroblasts or another mesodermal cell derivative. It is preferred thatthe feeder cells are human, non-human primate, pig, rat, or mouse feedercells, but any mammalian, avian, reptilian, amphibian, or piscine feedercells are acceptable. It is a yet more preferred embodiment that thefeeder cells be embryonic cells, although feeders from neonatal or adulttissue are acceptable. It is a yet more preferred embodiment that thefeeder cells be cloned and selected for the ability to support hepaticstem and progenitor cells. It is a still more preferred embodiment ofthe invention that hepatic stem and progenitor cells be cultured underclonal growth conditions, thereby permitting identification as hepaticcells and expansion of a population of clonal origin.

One preferred embodiment of the invention comprises mammalian hepaticprogenitor cells that are classical MHC class I negative and ICAM-1positive. A two color sort is a convenient method to isolate thebipotent cells: ICAM-1 positive and classical MHC class I negative aretwo parameters to define these cells. ICAM-1 positive cell populationsincludes hematopoietic, mesenchymal, and mature hepatic cells. Thedegree of expression is quite variable depending upon the status of thecells (for example, it is different in cells in an activated orquiescent state). Classical MHC class I antigen is expressed on allnucleated hematopoietic cells from stem cells to mature cells and onmature hepatocytes (although mature hepatocytes have less expressionthan hematopoietic cells). In rat fetal liver, classical MHC class Inegative cells include: bipotent hepatic progenitors, enucleated matureerythrocytes, and an unidentified cell population. In addition, thecells can express nonclassical MHC class I. Furthermore, the progeny ofprogenitors can express alpha-fetoprotein, albumin, or CK19 and can alsoexhibit a growth characteristic in which the cells grow in piles on topof each other, that is, in clusters.

It is an embodiment of the invention that the isolated progenitor cellshave the capability to divide and produce progeny. It is furtherpreferred that the progenitor cells are capable of more than about tenmitotic cycles. It is still more preferred that the progeny areprogenitor cells or hepatocytes and biliary cells. It is a preferredembodiment of the instant invention that isolated hepatic stem andprogenitor cells be committed to a hepatocyte or biliary cell lineage bythe selective application of Epidermal growth factor (EGF).

In a preferred embodiment, the process involves selecting for cells thatadditionally express alpha-fetoprotein and bind antibody specific foralpha-fetoprotein. In another preferred embodiment, the process involvesselecting for cells that, in addition, synthesize albumin and bindantibody specific for albumin.

It is a still more preferred embodiment of the instant invention thatisolated stem and progenitor cells be used as a component of anextracorporeal liver. It is a further more preferred embodiment of theinstant invention that the extracorporeal liver having isolated stem andprogenitor cells and their progeny be used to support the life of apatient suffering from liver malfunction or failure.

The invention discloses particular culture conditions that are requiredfor the ex vivo expansion of hepatic progenitor cells, here demonstratedfrom fetus. The inventors selected sublines of STO mouse embryonic cellsthat proved ideal as feeder cells. The feeder cells were used incombination with a novel, serum-free, hormonally defined medium (HDM).The combination enabled the inventors to establish various rat fetalhepatic cell lines from E15 liver in the rat without malignanttransformation of the cells. The inventor discloses the use of thehepatic cell lines and the HDM-STO co-culture system for development ofan in vitro colony forming assay (CFA) for defining clonal growthpotential of hepatic progenitors freshly isolated from liver tissue. TheCFA, when combined with cells sorted by a defined flow cytometricprofile, reveals bipotent hepatic progenitors. For example progenitorsfrom E13 rat livers, corresponding to E11.5 in the mouse, and with highgrowth potential have the phenotype as negative for classical MHC classI (RTIA region in the rat), dull positive for OX18 (monomorphic epitopeon MHC class I antigens), and ICAM-1 positive. The phenotype of RTIAnegative and OX18 dull positive is equivalent to nonclassical MHC classI (MHC class lb) dull positive. EGF is disclosed in this invention toinfluence both growth of the progenitor colonies and their fates aseither hepatocytes or biliary epithelial cells.

EXAMPLES

Glossary

Classical MHC class I antigen. The group of major histocompatabilityantigens commonly found mostly on all nucleated cells although they aremost highly expressed on hematopoietic cells. The antigen is also knownas MDHC class Ia. The nomenclature of the classical NHC antigens is afunction of species, for example in humans the MHC antigens are termedHLA. Table 3 provides nomenclature of classical MHC antigens in severalspecies.

Non classical MHC class I antigen. The group of major histocompatabilityantigens, also known as MHC class lb. that can vary even within aspecies. The nomenclature of the nonclassical MHC antigens varies byspecies, see, e.g., Table 4.

ICAM Intercellular adhesion molecule-1 (CD54) is a membrane glycoproteinand a member of the immunoglobulin superfamily. The ligands for ICAM-1are the β2-integrin, LFA-1 (CD11a/CD 18) and Mac-i (CD11b/CD18). Thismolecule is also important for leukocyte attachment to endothelium. Inaddition ICAM-1 has a role in leukocyte extravasation. The term ICAM-1is used to designate the form of these molecules found in mammals. Theterms ICAM or ICAM-1-like are used to designate the homologous andfunctionally-related proteins in non-mammalian vertebrates.

Debulking. Debulking is a process of removing major cell populationsfrom a cell suspension. In fetal liver the major non-hepatic lineagecells are red blood cells, macrophages, monocytes, granulocytes,lymphocytes, megakaryocytes, hematopoietic progenitors and stromalcells.

Dull positive. In fluorescence-activated cell sorting the intensity ofemitted light is proportional to the number of fluorochrome-conjugatedimmunoglobulin molecules bound to the cell which, in turn, isproportional to the density of the cell surface antigen under study. Asthe surface density or intracellular density of antigens can vary from afew to hundreds of thousands per cell, a wide range of fluorescenceintensities can be measured. The value of dull positive (or dull) isempirically determined and intermediate between the intensity ofbright-fluorescing cells with many antigens and dim cells with lowexpression of the specified antigen. The intensity may also be definedin terms of gates or intensity intervals. The dull positive phenotype isa feature of a weakly expressed antigen. The phenotype is also describedas weak or low expression.

Clonal growth. In cell culture, clonal growth is the repeated mitosis ofone single initial cell to form a clone of cells derived from the oneparental cell. The clone of cells can expand to form a colony or clusterof cells. Clonal growth also refers to the conditions necessary tosupport the viability and mitosis of a single cell. These conditionstypically include an enriched and complex basal nutrient medium, anabsence of serums, presence of specific growth factors and hormones,substrata of extracellular matrix of defined chemistry, and!orco-cultures of cells that supply one or more of the growth factors,hormones or matrix components.

Terms of enrichment. The term “remove” means to separate, select and setaside either to retain or discard. Thus, stromal cells can be removedfrom a mixed population by any of several means with the intent ofeither keeping them or of discarding them. The term “isolate” means toseparate from a larger group and keep apart. Thus, progenitor cells canbe isolated from a mixed population of progenitor and non-progenitorcells. The term “purify” means to separate away unwanted components.

Cluster growth. Hepatic progenitor cells frequently exhibit adistinctive feature, in which the cells divide and remain in mutualproximity. The progenitor cells form clusters in which cells are piledup one on another. Cells in the three-dimensional mass of piled-up cellsare adjacent to feeder cells or to other progenitor cells. The clustersare also termed P-colonies or P-type colonies and are distinct from cellmonolayers.

The following examples are illustrative of the invention, but theinvention is by no means limited to these specific examples. The personof ordinary skill in the art will find in these examples the means toimplement the instant invention. Furthermore, the person of ordinaryskill in the art will recognize a multitude of alternate embodimentsthat fall within the scope of the present invention.

Preparation and Analysis of Hepatic Stem and Hepatic Progenitor Cells

Rats. Pregnant Fisher 344 rats are obtained from Charles River BreedingLaboratory (Wilmington, Mass.). For timed pregnancies, animals are puttogether in the afternoon, and the morning on which the plug is observedis designated day 0. Male Fisher 344 rats (200-250 g) are used for adultliver cells.

Establishment of hepatic cell lines from embryonic day 15 livers. Fetallivers are prepared from day 15 of the gestation. Single cellsuspensions are obtained by incubating the livers with 0.05% trypsin and0.5 mMEDTA or 10 units/ml thermolysin (Sigma, St. Louis, Mo.) and 10units/ml deoxyribonuclease I (Sigma) for at 37° C. The cells areoverlayed on Ficoll-paque (Pharmacia Biotech, Uppsala, Sweden) forgradient density centrifugation at 450 g for 15 min. The cells from thebottom fraction are inoculated into tissue culture dishes coated with 17mg/ml collagen type IV (Collaborative Biomedical Products, Bedford,Mass.) or 12 μg/ml laminin (Collaborative Biomedical Products) forthl120-3 and rter6 or rhel4321, respectively. The serum-free hormonallydefined culture medium, HDM, is a 1:1 mixture of Dulbecco's modifiedEagle's medium and Ham's F 12 (DMEM/F12, GIBCO/BRL, Grand Island, N.Y.),to which is added 20 ng/ml EGF (Collaborative Biomedical Products), 5μg/ml insulin (Sigma), 10⁻⁷M Dexamethasone (Sigma), 10 μg/mliron-saturated transferrin (Sigma), 4.4×10⁻³M nicotinamide (Sigma), 0.2%Bovine Serum Albumin (Sigma), 5×10⁻⁵M 2-mercaptoethanol (Sigma), 7.6μeq/l free fatty acid, 2×10⁻³M glutamine (GIBCO/BRL), 1×10⁻⁶M CuSO₄,3×10⁻⁸M H₂SeO₃ and antibiotics. Each concentration given is the finalconcentration in the medium. After 4 weeks of culture, trypsinized cellsare cultured on a feeder layer of mitomycin C-treated STO mouseembryonic fibroblast line (American Type Culture Collection, RockvilleMd.). TH1120-3, rter6, and rhel4321 are cloned from three independentpreparations of fetal hepatic cells and are maintained on STO feedercells with HDM. After the establishment of the cell lines, theconcentration of EGF is reduced to 10 ng/ml for all cell cultures.

Dissociation of E13 of fetal liver. Fetal livers are dissected intoice-cold Ca++free HBSS with 10 mM HEPES, 0.5 mM MgSO₄ and 1 mM EGTA(pH7.4). The livers are triturated with 0.2% type IV collagenase (Sigma)and 16.5 units/ml thermolysin (Sigma) in HBSS prepared with 10 mM HEPES,0.8 mM MgSO4, and 1 mM CaCl₂. After incubation at 37° C. for 10 min, thecell suspension is digested with 0.025% trypsin and 2.5 mM EDTA (Sigma)for 10 min. Trypsin is then quenched by addition of 1 mg/ml trypsininhibitor (Sigma). Finally, the cells are treated with 200 units/mldeoxyribonuclease I (Sigma). In all experiments, 3-5×10⁵ cells per liverare obtained.

Isolation of adult liver cells. The two step liver perfusion method isperformed to isolate liver cells. After perfusion, the cells arecentrifuged for 1 min at SOg twice to enrich for large parenchymalcells. Cellular viability is >90% as measured by trypan blue exclusion.

Cell adhesion assay. Adhesion of cells to fibronectin (CollaborativeBiomedical Products), laminin and collagen type IV is evaluated using 96well micro-titer plates (Corning, Cambridge, Mass.) coated with theseproteins at 0.3 to 10 μg/ml. After removing the STO cells by Percoll(Pharmacia Biotech) gradient density centrifugation at 200 g for 15 min,3×10⁴ cells of the hepatic cell lines, thl120-3, rter6, and rhel4321,are cultured in each well for 10 hours with HDM. After rinsing twice toremove floating cells, fresh medium with the tetrazolium salt WST-1(Boehringer Mannheim, Indianapolis, Ind.) is added to measure the numberof variable adherent cells. After 4 hours, the absorbance is determinedaccording to the manufacturer's protocol.

STO Sublines. One hundred cells of parent STO from ATCC are cultured in100 mm culture dishes for 7 days in DMEM/F12 supplemented with 10%heat-inactivated fetal bovine serum, 2×10⁻³M glutamine, 5×10⁻⁵M2-mercaptoethanol and antibiotics. Four subclones are selected forfurther characterization according to the cell morphology and the growthspeed. Although CFA for rter6 is performed in the four subclones, one ofthem, STO6, does not persist in attaching to culture plates aftermitomycin C-treatment. One subclone, STO5, is transfected withpEF-Hlx-MClneo or pEF-MClneo kindly provided from Dr. J. M. Adams, TheWalter and Eliza Hall Institute of Medical Research. Linearized plasmidsat Nde I site are introduced into cells by DOSPER liposomal transfectionreagent (Boehringer Mannheim). After G418 selection, six clones areisolated. Three clones of each are analyzed by CFA.

Immunohistochemical Staining of Colonies. Culture plates are fixed inmethanol-acetone (1:1) for 2 min at room temperature, rinsed and blockedby Hanks Balanced Salt Solution (HBSS) with 20% goat serum (GIBCO/BRL)at 4° C. For double immunohistochemistry of alpha-fetoprotein andalbumin, plates are incubated with anti-rat albumin antibody (ICNBiomedicals, Costa Mesa, Calif.) followed by Texas Red-conjugatedanti-rabbit IgG (Vector laboratories, Burlingame, Calif.) andFITC-conjugated anti rat alpha-fetoprotein polyclonal antibody (NordicImmunology, Tilburg, Netherlands). For double labeling of albumin andCK19, anti-CK19 monoclonal antibody (Amersham, Buckinghamshire, England)and FITC-conjugated anti mouse IgG (Caltag, Burlingame, Calif.) are usedinstead of anti alpha-fetoprotein antibody.

Flow cytometric analysis. Cells are analyzed on a FACScan(Becton-Dickinson, Mountain View, Calif.) and sorted using a Moflow FlowCytometer (Cytomation, Fort Collins, Colo.). The cell suspensions fromE13 fetal liver are incubated with HBSS, containing 20% goat serum(GIBCO/BRL) and 1% teleostean gelatin (Sigma), on ice to preventnonspecific antibody binding. After rinsing, the cells are resuspendedwith FITC-conjugated anti rat RT1A^(a,b,1) antibody B5 (Pharmingen, SanDiego, Calif.) and PE-conjugated anti-rat ICAM-i antibody 1A29(Pharmingen). In some experiments the cells are stained withbiotinylated anti-rat monomorphic MHC class I antibody OX 18(Pharmingen) followed by a second staining with streptavidin-red670(GIBCO/BRL) for 3 color staining. All stainings are performed withice-cold Ca++ free HBSS containing 10 mM HEPES, 0.8 mM MgSO4, 0.2 mMEGTA, and 0.2% BSA (pH7.4). The established three hepatic cell lines aretrypsinized and fractionated by Percoll density gradient centrifugationto remove feeder cells. The rat hepatoma cell line, FTO-2B, and the ratliver epithelial cell line, WB-F344, as well as adult liver cells arestained to compare with the fetal hepatic cell lines. The cell lines arekind gifts of Dr. R. E. K. Fournier, Fred Hutchinson Cancer ResearchCenter, Seattle, Wash., and Dr. M. -S. Tsao, University of NorthCarolina, Chapel Hill, N.C., respectively. Cells are blocked and stainedwith FITC conjugated B5, OX 18, PE-conjugated 1A29 or antiFITC-conjugated rat integrin β1 antibody Ha2/5 (Pharmingen).FITC-conjugated anti mouse IgG is used for OX18. Cell suspensions ofthree fetal hepatic cell lines are stained with biotinylated anti-mouseCD98 followed by a second staining with streptavidin-red670 as well asanti-rat moAb to gate out mouse cell populations.

CFA for hepatic cell lines sorted cells, and adult liver cells. Thehepatic cell lines are plated in triplicate at 500 cells per 9.6 cm² onmitomycin C-treated STO feeder layer with the same HDM as used formaintaining each cell line. Before plating, cell are trypsinized andfractionated by Percoll density gradient centrifugation to remove feedercells. The cultures are incubated for 10 to 14 days with medium changesevery other day. Double immunofluorescence staining of alpha-fetoproteinand albumin is then performed. 100 colonies per well are analyzed by thecolony morphology, P or F type, and the expression of alpha-fetoproteinand albumin. The colonies are stained using Diff-Quick (Baxter, McGawPark, Ill.) to count the number of the colonies per well. In the CFA forprimary sorted cells and adult liver cells, the plating cell number ischanged as described. As another minor modification, the culture periodis expanded to between 14 and 17 days, and the concentration ofdexamethasone is increased to 10⁶M. All other procedures are performedas above. In the CFA for adult liver cells, small numbers of clumps ofliver cells are not eliminated from the cell suspension after thepreparation. Therefore, an undefined number of the colonies might beproduced from the clumps. For CFA of biliary differentiation on sortedcells, double immunofluorescence staining of albumin and CK19 of thecolonies is performed at 5 days each of the culture in the presence orabsence of EGF. At day 5 of the cultures, any colony with more than oneCK19⁺ cell is counted as a CK19⁺ colony. At day 10 and 15, coloniescontaining multiple clusters of two CK19⁺ cells or one cluster of morethan three CK19⁺ cells are counted as a CK19⁺ colony. About 100 coloniesper well are counted. Each point represents the mean ±SD fromtriplicate-stained cultures.

Generation and Characterization of Fetal Rat Hepatic Cell Lines UsingFeeders Of Mouse Embryonic Cells with a Hormonally Defined Medium.

Simple long-term cultures of rat E15 hepatic cells are attempted to seehow long fetal hepatic cells could be maintained and expanded ex vivo toproduce progeny. After a gradient density centrifugation to removehematopoietic mononuclear cells, the fetal liver cells are cultured onculture dishes coated by collagen type IV or laminin and in HDM (seeexample 6.1). The cells survive well for more than 4 weeks. However,secondary cultures on fresh collagen type IV- or laminin-coated dishesdo not permit further expansion. When mitomycin C-treated STO embryonicmouse fibroblast cell lines are used as a feeder layer for the secondarycultures, many aggregates of cells grow. Eventually several stablehepatic cell lines are established from four independent experiments.

Immunohistochemical analysis of alpha-fetoprotein and albumin areperformed in the continuous growing cell populations before cloning ofthe cell lines. Both proteins, alpha-fetoprotein and albumin, are usedas the markers to confirm that cell populations originated from thehepatic lineage. A cell population with a tendency to form piles ofcells, called P-colonies, had intense expression of alpha-fetoproteinand albumin, while another cluster produced flattened monolayers, calledF-colonies, with diminished expression of alpha-fetoprotein and noalbumin. The embryonic mouse fibroblasts, STO, do not show anyreactivity to either antibody. For further analysis, three clonedhepatic cell lines from independent experiments are selected by themorphological criteria of either P type or F type colonies (FIG. 1A-1C).Rhe14321 (FIG. 1A) consists mostly of packed small cells, P typecolonies, whereas th1120-3 (FIG. 1C) makes only a flattened monolayer ofF-type colonies. Rter6 (FIG. 1B) is an intermediate phenotype of thesetwo. Interestingly, the heterogeneity of rter6 is still observed afterthree rounds of sequential cloning of the flattened colony. To see theheterogeneity of colonies derived from single cells in rhel4321 andrter6, the cells are cultured on STO fibroblasts for 10 to 14 days at aseeding density of 500 cells per 9.6 cm² (one well of a 6-well plate).The colonies are then characterized in terms of their morphology andtheir expression of alpha-fetoprotein and albumin. FIG. 2A-2F shows theresults. In the cell lines, rhel4321 (FIG. 2B) and rter6 (FIG. 2C), andin the original cell population prior to cloning (FIG. 2A), almost allP-type colonies strongly express alpha-fetoprotein, whereas F-typecolonies of cells do not. Furthermore, the intense expression of bothalpha-fetoprotein and albumin is observed only in P type colonies. Themorphological difference in the cloned hepatic cell lines correlate tothe percentage of the P type colony (FIGS. 2B and 2C). The percentage ofP type colonies in CFA of rter6 and rhel4321 is 33.3% (±8.6% SD) and65.7% (±4.0% SD), respectively. The total colony number per well iscounted to calculate the clonal growth efficiency (colony efficiency).The efficiency of rter6 and rhel4321 is 45.7% (±1.3% SD) and 36.4%(±1.1% SD), respectively. The th1120-3 cells tightly attach to eachother along their lateral borders making preparation of single cellsuspensions difficult. However, the th1120-3 cells do not produce piledup clusters (FIG. 1C).

Next, the preferences of each of the cell lines to adhere to specificcomponents of extracellular matrices (ECM) are tested, because theadhesion of mouse liver cells to such ECM proteins as laminin, collagentype IV, and fibronectin, changes in different developmental stages.Whereas collagen type IV is the most effective in the attachment ofth1120-3 (FIG. 1C), similar to the findings for the adult liver cells,it works less well for rter6 (FIG. 1B) and rhel4321 (FIG. 1A). Lamininis the most effective substratum for adhesion of rhel4321 (FIG. 1A).This preference is similar to that of primary cultures of mouse fetalliver cells (Hirata et al., 1983). In summary, the conserved expressionof alpha-fetoprotein and albumin in P-type colonies and preferentialadherence to laminin by rhel4321, suggest that the cell populationsproducing P type colonies are more strictly associated with hepaticprogenitor cells.

Isolation of STO Subclones for the Colony Formation; Assay of HepaticProgenitors

To develop a CFA system to identify bipotent hepatic progenitors withhigh growth potential, the culture system has to be able to support cellexpansion at clonal seeding densities and with conservation of criticaloriginal hepatic functions. Albumin and alpha-fetoprotein are two of themost significant markers for early hepatic development. The cultureconditions optimizing P type colonies should be the best, since P type,but not F type, colonies maintain the expression of alpha-fetoproteinand albumin during clonal expansion. Therefore, STO subclones arecompared in their support of P type colonies of rter6. One of theclones, STO5, supports the P type colony formation more than any of theother sublines and more than the parent line (FIG. 2D). The CFA ofrhel4321 also confirms that STO5 is a more effective feeder than theparent STO (FIG. 2E). The mouse Hlx gene product, expressed in themesenchymal cells lining digestive tract from E10.5, is essential forfetal hepatic cell expansion. Although the mRNA expression for the Hlxgene is analyzed in all the STO subclones, there is no significantdifference in its expression among the subclones (data not shown).Furthermore, the stable transfectants of mouse Hlx in STO5 do not resultin an improvement in the colony formation assays (FIG. 2F). One clone ofthe transfectants, however, is used for further experiments, because thetransfectant supports a more stable persistence of the originalmorphology of STO5 at relatively high passages.

Identification of Hepatic Progenitors from E13 Fetal Liver using theSurface Antigenic Markers and the Colony Forming Assay.

Hepatopoiesis and massive amounts of hematopoiesis co-exist in the fetalliver. So far, the antigenic profile of hematopoietic progenitors hasextensively been analyzed, whereas studies of early hepatic progenitorsare still in their infancy. The antigenic profile of hepatic cells isanalyzed using the three hepatic cell lines established in this study,an adult hepatocarcinoma cell line (FTO-2B), an epithelial cell linefrom adult rat liver (WB-F344), and freshly isolated adult liver cells(FIG. 3A-3X). Compared with FTO-2B, WB-F344, and adult liver cells, thepattern of the most immature of the fetal hepatic cell lines, rhel4321,is quite unique in that there is no expression of classical MHC class I(RT1A¹) (FIG. 3A). The cell line th1120-3 is similar to rhe14321 in thepattern of RT1A¹ (FIG. 3I), OX18 (pan-MHC class I) (FIG. 3J), and ICAM-1(FIG. 3K), whereas rter6 has relatively high expression of RT1A¹ (FIG.3E) and OX18 (FIG. 3F). Additionally, another cell line from a differentexperiment, which has an identical morphology to rhel4321 (FIG. 1A-1D),is also RT1A¹⁻, OX18^(dull), and ICAM-1⁺. Integrin b₁ expression issimilar in all the cell lines, while the pattern of RT1A^(a b,1) andICAM-1 is unique among them. The antigenic profile of adult liver cellsis RT1A¹⁺ (FIG. 3U), OX18⁺ (FIG. 3V), and ICAM-1+(FIG. 3W). Since, inthe adult rat, all bone marrow cells except mature erythrocytes stronglyexpress MHC class I molecules, the fetal hepatic population can beseparated from the hemopoietic cell populations by MHC class Iexpression. The cell suspensions from rat E13 livers are stained withanti RT1A¹ and ICAM-1 antibodies. FIG. 4A 1 to 4A2 shows the 2color-staining pattern of RT1A¹ and ICAM-1. To determine which fractioncontains the hepatic cell population, five fractions (FIG. 4B-1 to 4B-5)are isolated by fluorescent activated cell sorting and then screened byCFA for clonal growth potential. FIG. 4B-1 to 4B-5 represents the resultof resorting of the five fractions after sorting. The hepatic cellcolonies, defined by expression of albumin and alpha-fetoprotein, aredistinguishable also morphologically, enabling one to count the numberof hepatic colonies per well. The majority of the hepatic colonies aredetected in the gate RT1A^(1dull) and ICAM-I⁺ (Table 1, FIG. 4B-2, i.e.gate 2), and the frequency of the P type colony is 75.6% (±4.9% SD).Gate 1 (FIG. 4B-1) shows a much lower number of the colonies, and theother fractions contain negligible numbers of cells with colony formingability. In gates 1 and 2, the expression of both alpha-fetoprotein andalbumin is confirmed in all the hepatic colonies. Some of the colonies,derived from cells in gate 2, are larger than others. To investigate theMHC class I expression on the hepatic cells in detail, three colorstaining of RT1A¹, ICAM-1, and OX18 with the sidescatter (SSC) asanother parameter is used for the cell fractionation. Sidescatter (SSC),a reflection of the granularity of cell, is a useful parameter forseparation of hepatic from hematopoietic cells, because fetal hepaticcells contain lipid droplets as early as E11 of gestation. FIG. 4C-1 to4C-5 shows that the gate 2 contains the highest number of colony-formingcells. Gating R2 based on the SSC, the population corresponding to thegate 2 clearly shows RT1A¹⁻ and OX18^(dull) phenotype (FIG. 4C-1 to 4C-5and FIG. 4D-1 to 4D-4). The CFA confirms that R4 harbors morecolony-forming cells than gate 2 (Table 1). These results suggest thatmost of the RT1A¹⁻, OX18^(dull), and ICAM-1⁺ population from E13 ratliver are hepatic cells producing alpha-fetoprotein⁺ and albumin⁺colonies. It is the identical antigenic profile found for rhel4321 cells(FIG. 3A to 3D). TABLE 1 The Frequency of hepatic colonies from sortedE13 fetal liver based on the expression of RT1A and ICAM-1. InoculatedHepatic Efficiency of Gate cell (per well) colony (per well) colonyformation (%) 1 1000 8.7 ± 4.0 0.87 2 500 136.3 ± 4.6  27 3 5000 10.0 ±7.9  0.13 4 5000 6.3 ± 0.6 0.13 5 5000 5.0 ± 1.0 0.10 R3 1000 7.0 ± 2.60.70 R4 500 269.3 ± 9.8  54

Colony forming culture on STO5hlx containing indicated cell number fromeach fraction of E13 of fetal liver. Number of the hepatic colonies wasestablished from triplicate stained cultures (mean±SD). Efficiency ofthe colony formation express the percentage of cells inoculated toculture that went on to form colonies analyzed after 16 days of theculture.

Different Growth Requirement of E13 Hepatic Cells and Adult Liver Cells

The growth requirement of the sorted hepatic cells from E13 liver arestudied using the defined STO5 feeders and the HDM. EGF has long beenknown as a potent growth factor for adult liver cells. Therefore, theeffects of EGF for colony formation of sorted hepatic cells areinvestigated. The colony-size of the RT1A¹⁻ OX18^(dull), ICAM-1⁺ hepaticcells becomes bigger in the absence of EGF, whereas adult liver cellsyielded colonies only in the presence of EGF (FIG. 5A). Furthermore, themorphology of the colonies derived from adult liver cells is the typicalF type, whereas all RT1A¹⁻ hepatic cells produce P type colonies withoutEGF. However, the colony efficiency is reduced slightly by the absenceof EGF (FIG. 6A). Interestingly, the culture condition in the absence ofEGF emphasized the two types of P-colonies, P1 and P2. Although themajority of the colonies is P2 type, at the 12th day of culture, it isdifficult to distinguish the two types definitively because some of themdo not have the typical morphology. These results suggest that fetalhepatic cells and adult liver cells are intrinsically different in theirgrowth requirement as well as in their expression of RT1A¹ (FIGS. 3 and4) and colony morphology.

After 3 weeks of culture, when growth seems to reach a maximum, theexpression of RT1A¹⁻, OX18, and ICAM-1 is assessed. As shown in FIG. 5Bto 5D, the expression of RT1A¹ is not induced, while that of OX18 isreduced. The level of ICAM-1 does not change. Furthermore, the averagecell number of single colony is calculated from the recovered cellnumber, the percentage of rat hepatic cells and the colony efficiency.The estimated cell number reaches 3 to 4×10³ (Table 2). This indicatesthat the single cell forming the colonies divided approximately 11-12times on average under this culture condition. TABLE 2 Calculation ofthe cell number in single hepatic colony. Average of cell Inoculatedcell Seeding density Culture length Recovered Percentage of Colonynumber in number (cell/cm²) (day) cell number rat cell (%) efficiency(%) single colony 500 18 18 1.5 × 10⁶ 58 41 4.2 × 10³ 4000 51 21 6.0 ×10⁶ 90 44 3.1 × 10³ 4000 51 20 4.0 × 10⁶ 69 21 3.3 × 10³

Sorted cells from R4 in FIG. 4C-5 were cultured on STO5hlx feeder cellsin 60 mm or 100 mm dish. After the period indicated of the culture cellall cells were recovered and the total cell number counted. Thepercentage of rat cells is from flow cytometric analysis based on theexpression of rat ICAM-1 and mouse CD98. Colony efficiency indicates thepercentage of cells inoculated to culture that went on to form colonies.Data from triplicate-stained cultures (mean) was obtained from theexperiments run parallel with. Average of cell number in singlecolony=(Recovered cell number ×Percentage of rat cell/100)/Inoculatedcell number×Colony efficiency/100)

Evidence for Bipotentiality in RT1A¹⁻ Hepatic Progenitors

At E13 of gestation in the rat, the hepatic cells are thought to have abipotent precursor giving rise to the mature hepatocyte and bile ductepithelium. However, before the discoveries of the instant inventionthere has been no direct evidence whether the two fates originated froma single cell or not. To determine whether the RT1A¹⁻ OX18^(dull)ICAM-1⁺ fetal hepatic cells can differentiate to the biliary lineage inthis culture system, the colonies are stained by anti-CK19 as a specificmarker for biliary epithelial cells. CK19 is expressed in the bile ductepithelial precursors after day 15.5 in the fetal rat liver at whichtime the expression of albumin disappears in the cells. The sortedRT1A¹⁻ ICAM-1⁺ cells are cultured in the presence or absence of EGF, andtheir fates are monitored by the expression of CK19 and albumin after 5days of culture. After the first 5 days, the CK19⁺ colonies arenegligible in the cultures treated with EGF, whereas a few coloniescontaining CK19⁺ cells occurred in those in the absence of EGF (FIG. 6Ato 6B). Although the intensity of the CK19 expression is fairly weak,the CK19⁺ cells show reduced albumin expression. At the 10th day of theculture, as shown in FIGS. 6A to 6B, some colonies apparently expressonly CK19 or albumin and others have dual positive expression. Thepattern of the CK19⁺ and albumin⁺ cells in a single colony isreciprocal. The number of dual positive colonies and CK19 singlepositive colonies still is higher in the absence of EGF (FIG. 6A). Inthe presence of EGF, many of the colonies consist only of albumin⁺ cellsat the 10th day (FIG. 6B). Eventually, the percentage of dual positivecolonies reaches nearly 100% in the absence of EGF at day 15 (FIG. 6A).Altogether, EGF dramatically suppresses the appearance of CK19⁺ coloniesthrough the culture (FIG. 6B). These results suggest that the RT1A¹⁻,OX18^(dull), and ICAM-1⁺ cells from E13 fetal liver can differentiatetowards the biliary lineage and their fate can be influenced by EGF invitro (FIG. 7).

Isolation of Human and Non-Human Hepatic Precursors using Antibodies toICAM and Classical MHC Class I Epitopes.

The molecular structure and biological function of classical MHC class Iantigens are highly conserved among vertebrates, and the same is thecase for the ICAM antigens. However MHC antigens are not found ininvertebrates. MHC antigens are the most comprehensively investigatedmolecules of vertebrate species. Although the information on ICAMantigens is limited, the biological functions of ICAM antigens areconserved in many mammals such as human mouse, and rat. So far, ICAM-Icomplementary DNA has been cloned from human, chimpanzee, mouse, rat,dog, and bovine. The conclusion from the sequence data is that themolecular structure is highly conserved in all species. Therefore, bychoosing antibodies specific for the ICAM-1 in a given species andantibodies for the designated class I MHC antigen according to thetable, the cell populations enriched in hepatic progenitor cells can beisolated. TABLE 3 Major Histocompatability Antigens Nomenclature SpeciesRats Mice Humans MHC RT1 H-2 HLA Classical MHC class I A K, D, L A, B, CNonclassical MHC C/E, M TL, Q, M E, F, G, H, J, X class I

OX18 recognizes a monomorphic epitope of rat MHC class I antigens.Therefore, the antibody recognizes nonclassical MHC class I as well asclassical MHC class I. The exact number of nonclassical MHC class I lociare not defined in any species, because it varies between members of thesame species. Therefore, in the future, a new locus might be discoveredas a nonclassical MHC class I in subpopulations of these species. Oneembodiment of the invention is a method of predicting the phenotype ofhepatic progenitor cells. This feature is illustrated in the table ofkey cell surface markers in various species. TABLE 4 Markers for HepaticProgenitor Cells, based on the Instant Invention. Species Rats MouseHuman Classical RT1A- H-2K negative and/or HLA-A negative MHC class 1Negative H-2D negative and/or and/or HLA-B H-2L negative and/or Cnegative Nonclassical Dull positive Dull positive for TL Dull positivefor MHC class I for C/E and/ and/or Q and/or M E, F, G, H, J or Mand./or X ICAM-1 Positive Positive Positive

Characterization of Rat Bipotent Hepatic Progenitors and Comparison withAdult Hepatocytes TABLE 5 Cell Surface and Internal Markers in RatCells. Markers Bipotent Hepatic Cells Adult Hepatocytes** Data FromFreshly Isolated Cells ICAM-1 + + CD90 (Thy-1) − − CD44H + −* Class 1MHC − + (RT1A¹) OX18 Dull + Data From Cultured Cells Alpha-fetoprotein ++in several of the cells in most colonies Albumin +EGF: many cellspositive + −EGF: fewer cells positive CK19 +EGF: few cells positive −***−EGF: many cells positiveEGF = epidermal growth factor that when added to the culture conditionsappears to drive the cells towards the hepatocytic lineage and blocksdevelopment of the biliary lineage. In the absence of EGF, there isspontaneous differentiation towards both biliary and hepatocyticlineages.*Others have shown that adult hepatocytes and adult biliary epitheliaare negative for CD44H (Cruishank SM et al, J Clin Pathol 1999 52:730-734) and CD 90 (Gordon G et al American Journal of Pathology 157:771-786).**Adult hepatocytes are those that can proliferate by hyperplasticgrowth in culture under the conditions specified above.***CK19 is not expressed on adult heptocytes in vivo. However, in anyculture of adult liver cells, one can observe one or two cells thatexpress some CK19 but without apparent inducibility by cultureconditions and without distinctions morphological between the positivecells. This is in contrast to the observations in fetal liver in vivoand in the cultures of bipotent cells and of other fetal liver cells.

Antigenic Phenotyping of Human Fetal Liver Cells

Human fetal liver cells are stained with antibody to CD14. Severalpopulations are identified by two-color cell sorting of HLA (ABC) vs.CD14. These populations include a group designated R2 characterized byintermediate HLA staining and without CD14 staining and another groupdesignated R3 characterized by high CD14 staining and high HLA staining.When stained for alpha-feto protein, the R3 cells are positive foralpha-fetoprotein and the R2 contains two subpopulations, only one ofwhich stains for AFP.

Further Isolation of Human Hepatic Precursors Using Antibodies toExpression Markers Including Nonclassical MHC Class I,Alpha-Fetoprotein, Albumin, and CKI 9.

In order to select monomorphic epitopes the cell suspension is incubatedwith fluorescein-conjugated antibody to the HLA class I monomorphicepitopes. The one skilled in the art will recognize that any of manyother fluoro chromes can be used in place of fluorescein, including, butnot limited to rhodamine and Texas Red. As an alternativeindirect-immuno fluorescence is used to label the cells. That is, thefluorescent label is conjugated to an antibody directed to theimmunoglobulin of the species in which the primary antibody is elicited.The cell sample is sorted by high throughput fluorescence-activated cellsorted using any of a variety of commercially available or customizedcell sorter instruments. Hepatic progenitor cells that have intermediateor dull fluorescence with the labeled anti-monomorphic epitopes areselected.

Compositions enriched in rat hepatic progenitors can also beadvantageously prepared by sorting liver cell suspensions usingantibodies to CD44H. Liver cells that show a high level of sidescatteralso express CD44H and express alpha fetoprotein. In particular, cellsthat express alpha-fetoprotein also express higher levels of CD44H. Incontrast, liver cells that have a low level of sidescatter do notexpress CD44 at higher levels.

Liver cells that show a high level of sidescatter do not show aCD90-dependent distinction in alpha-fetoprotein expression. However,cells that show a low level of sidescatter show a CD90-dependentdistinction in alpha-fetoprotein expression. In particular, the cellsthat express alpha-fetoprotein also express higher levels of CD90.

As an alternative, antibodies specific for polymorphic epitopes,including but not limited to, HLA-A2, HLA-B27, and HLA-Bw22, are used toidentify and isolate hepatic progenitors.

Furthermore, antibodies specific for nonclassical HLA class I antigens,including HLA-G, HLA-E, and HLA-F, are used to identify and isolatehepatic progenitor cell that express the antigen.

It is evident that these methods are readily adaptable to non-mammalianhepatic progenitor cells.

Further Isolation of Human Hepatic Precursors using High-ThroughoutAffinity Isolation Methods With Antibodies to Expression MarkersIncluding Alpha-Fetoprotein, Albumin, Nonclassical MHC Class I and CK19

An isolation protocol is presented in diagrammatic form as follows:

Other methods of debulking and eliminating the red blood cells componentcan be advantageously used and these methods can reduce some of thestromal cell population as well. These methods include fractionation onPercoll gradients and specific depletionusing antibody to glycophorin A,CD45, or both. Furthermore, these methods include sedimentationvelocity, separation in density gradients other than Percoll, e.g.,Ficoll, zonal centrifugation and cell elutriation. By these methods redblood cells, polyploidy hepatocytes, hemopoietic cells, and stromalcells are removed.

Isolation of cell populations that are positive for ICAM-1 and negativefor classical MHC class I antigen are further characterized with othermarkers including nonclassical MHC class I to identify hepaticprogenitors. In addition, the progeny of these progenitor cells labeledwith antibodies to the cytoplasmic proteins, such as alpha-fetoproteinand/or albumin, markers that are long-known to be characteristic ofhepatic progenitors. Alpha-fetoprotein and albumin are representative ofthe well known markers for hepatic progenitors that cannot be used toselect for viable cells, since labeling the cells for those proteinsrequires permeabilization of the cells, a process that destroys theirviability. However, cell samples from a population can be tested foralpha-fetoprotein, albumin, and cytokeratin. Thereby, thecharacteristics of the whole population are deduced. However, the highcorrelation between the cell surface markers (e.g., ICAM-1 positive,OX-18 dull positive, classical MHC class I negative) and clonal growthcapability with the cytoplasmic markers alpha-fetoprotein, albumin, orCK19 demonstrates that viable cells can be isolated using selection forthe surface markers alone.

Further Isolation of Human Hepatic Precursors using Sidescatter.

Side scatter cannot be used, by itself, to identify a cell type such asthe hepatic precursors. However, it is very useful as an adjunct toselection by other means such asfluorescence activated cell sorting formarkers. For a population identified by a given marker, such asclassical MHC class I, one must focus on a subpopulation defined bytheir side scatter characteristics (See FIG. 4 c).

It is important to realize that mature hepatic cells are highly granular(show very high side scatter); the hepatic progenitors are intermediatein granularity; and the non-parenchymal cell populations have even lessgranularity than the hepatic precursors. In cells from fetal tissue,consisting almost entirely of non-parenchymal cells and hepaticprogenitors, the hepatic progenitors have the highest granularity.Hepatic progenitors are selected as the cell population that isintermediate in granularity by flow cytometry.

Compositions enriched in human hepatic progenitors can also beadvantageously prepared by sorting liver cell suspensions usingantibodies to CD14 in combination with antibodies to HLA, the humanversion of MHC. All the methods of immunoselection are equallyapplicable. As a particular example, flow cytometry is used to isolatecells: cells designated R2 which express relatively intermediate levelsof HLA and do not express CD14, and cells designated R3 which expressrelatively high levels of HLA and relatively high levels of CD14. The R2cells are further characterized to have two subpopulations by expressionof alpha-fetoprotein. The R3 cells are further characterized to consistonly of cells that express alpha fetoprotein.

Removal of Non-Hepatic Progenitor Cells by Negative Selection withAntibodies to Glycophorin A or CD4S.

The hepatic progenitors are distinguished from red blood cells by use ofmonoclonal antibodies (Glycophorin A for human) and a polyclonalantiserum to red blood cell antigen if monoclonal antibodies are notavailable. Also, cells that express common leukocyte antigen (CD45) alsoexpress classical MHC class I antigen. Therefore, by default, CD45 isnot an antigen that can be used to identify the rodent hepaticprogenitor cells but is used as an alternative or supplement to thenegative selection by classical MHC class I.

Identification of Hepatic Cancers and Response to Treatment

The markers we have used to identify hepatic progenitors includingnonclassical HLA class I antigens, ICAM-1 and alpha-fetoprotein can beused to characterize liver cancers to better define successfultreatments of those cancers. Cancers, in general, are transformants ofstem cells and early progenitor cell populations. However, thesetransformants often retain expression of the antigenic markers sharedwith their normal counterparts. Liver cancers, distinguished by theseantigenic markers, can identify cancers responding in distinct ways tooncological therapeutic modalities (e.g., chemotherapeutic drugs,radiation, and adjuvant therapies).

Identification and Selection of Embryonic Stem Cells

The markers described here and the methodologies for selection can bealso be used to characterize the differentiation of embryonic stem (ES)cells to certain fates. ES cells are becoming popular as possibleall-purpose stem cells for use in reconstitution of any tissue. However,past studies of injection of ES cells into tissues resulted in tumors,some of which were malignant. The only way the ES cells are to be usedclinically is to differentiate them to determined stem cells and theninject the determined stem cells. Thus embryonic stem cells aremaintained in cell culture under culture conditions that permitproliferation to form progeny. The ES progeny are subjected to flowcytometry after incubation with antibodies to classic MHC class I andICAM-1 antigens. ES progeny meeting the criteria for hepatic progenitorsare expanded in cell culture. The markers we have identified can be usedto define an hepatic fate for a determined stem cell.

Use in Conjunction with Gene Therapy

The markers of liver progenitor cells identified here are used toidentify cell populations for gene therapies. To date, gene therapieshave often not worked or not worked well with targeting to mature cellpopulations. The major successes in gene therapies to date have been exvivo gene therapies in hemopoietic progenitor cell populations.Therefore, ex vivo gene therapies for liver are used withhepatic-determined stem and progenitor cells isolated by our protocols.Also, the gene therapies involving “targeted injectable vectors” areimproved by focusing on those that target hepatic progenitors. In theseways inborn errors of metabolism can be improved, including hemophilia,respiratory chain complex I deficiency, phenylketonuria, galactosemia,hepato-renal tyrosinemia, hereditary fructose intolerance, Wilson'sdisease, haemochromatosis, endoplasmic reticulum storage disease,hyperoxaluria type 1,3 betahydroxy-delta 5-C27-steroid dehydrogenasedeficiency, glycogen storage diseases (including deficiency ofglucose-6-phosphatase, glucose-6-phosphate translocase, debranchingenzyme, liver phosphorylase and phosphorylase-b-kinase), fatty acidoxidation or transfer defects (including organic acidurias, defects ofacyl-CoA dehydrogenases), porphyria, and bilirubin uridine diphosphateglucuronyltransferase.

Hepatic progenitors can be used for gene therapy as follows:

Phenylketonuria (PKU) is an autosomal recessive disorder caused by adeficiency of phenylalanine hydroxylase (PAH) in the liver. PAHcatalyzes the conversion of phenylalanine to tyrosine usingtetrahydrobiopterin as a cofactor. Patients with PKU show profoundmental retardation and hypopigmentation of skin, hair, and eyes due toincreased amount of phenylalanine in body fluids. Although the rigiddietary restriction significantly reduces serum phenylalanine levels,reduced compliance, even in adolescence or early adulthood, often leadsto a decline in mental or behavioral performance. Gene therapy techniqueis one alternative to dietary therapy for PKU. The development of amutant mouse Pah^(enu2) for PKU facilitated effects to attempt thisapproach. So far, three different vector systems, recombinantadenoviruses, retroviruses, and DNA/protein complexes have beendeveloped. The effect of adenovirus-mediated gene transfer lasted foronly short period after the injection because of the host immuneresponse against the recombinant virus. Although recombinantretroviruses and DNA/protein complexes can effectively transducePAH-deficient hepatocytes in vitro, the clinical utility of the ex-vivoapproach is limited primarily because of the low number of cells thatcan be successfully reimplanted into liver. Use of hepatic progenitorswith high growth potentiality can eliminate the problem mentioned above.

Use of Bipotent Hepatic Progentiors in Cell Therapy

A rat model of liver failure is used to evaluate heterogenous celltransplantation therapy. Liver failure is modeled by surgical removal ofabout 70% of the liver and ligation of the common bile duct in anexperimental group of ten male rats (125 to 160 g body weight). A shamcontrol group of ten age- and sex-matched rats is subjected to s similaranesthesia, mid-line laparotomy, and manipulation of the liver, butwithout ligation of the bile ducts and without hepatectomy.

An enriched population of hepatic precursors is prepared as describedabove. In brief, the livers of 12 embryonic (embryonic day 14) rat pupsare aseptically removed, diced, rinsed in 1 mM EDTA in Hank's BSSwithout calcium or magnesium, pH 7.0, then incubated for up to 20minutes in Hank's BSS containing 0.5 mg/ml collagenase to produce a nearsingle cell suspension.

Bipotent hepatic progenitors are prepared by any of the above methods.

On day three after the hepatectomy or sham operation, the rats, bothexperimental and sham control, are subjected to a 5 mm abdominalincision to expose the spleen. One half of each of the experimental andsham control group animals, randomly chosen, are injected with 01.1 mleach of the bipotent hepatic progenitors composition, directly into thespleen. All incisions are closed with surgical staples. The number ofcells administered to different groups of animals can be about 10³ up toabout 10¹⁰, in particular, 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰. Theimmunosuppressant cyclosporine A, 1 mg/kg body weight, is administereddaily intraperitoneally.

Blood levels of bilirubin, gamma glutamyl transferase and alanineaminotransferase activities are monitored two days before thehepatectomy or sham hepatectomy operation and on post-operation days 3,7, 14, and 28. Body weight, water consumption, and a visual inspectionof lethargy are recorded on the same days. At 28 days post hepatectomyall surviving animals are killed for histological evaluation of spleenand liver.

The above examples have been depicted solely for the purpose ofexemplification and are not intended to restrict the scope orembodiments of the invention. Other embodiments not specificallydescribed should be apparent to those of ordinary skill in the art. Suchother embodiments are considered to fall, nevertheless, within the scopeand spirit of the present invention. Thus, the invention is properlylimited solely by the claims that follow.

1. A method of obtaining a population of cells enriched in human hepaticprogenitors comprising: (a) obtaining a cell suspension of human livercells and (b) sequentially, in any order, or substantiallysimultaneously, (i) removing from the cell suspension those cells thatexpress at least one human MHC class Ia antigen, (ii) isolating from thecell suspension those cells that are positive for an ICAM antigen; and(iii) isolating from the cell suspension those cells that exhibit atleast one of the following characteristics: (1) expression of at leastone of CD44H, alpha-fetoprotein, albumin or CK19, or (2) dull expressionof a nonclassical human MHC class Ia antigen, or (3) higher side scatter(SSC) relative to non-parenchymal cells as measured in a flow cytometerto provide a population of cells enriched in human hepatic progenitors.2. A method for identification of human hepatic progenitor cells,comprising: (a) providing a liver cell suspension suspected of includinghuman hepatic progenitor cells; (b) identifying cells which express ICAMantigen and do not express MHC class Ia antigen; and (c) identifyingwhich cells exhibit at least one of the following characteristics: (1)expression of at least one of CD44H, alpha-fetoprotein, albumin or CK19,or (2) dull expression of a nonclassical human MHC class Ia antigen, or(3) higher side scatter (SSC) relative to non-parenchymal cells asmeasured in a flow cytometer.
 3. The method of claim 1 in whichisolation of those cells that exhibit a dull expression of nonclassicalhuman MHC class lb Ia antigen is carried out with an antibody againstOX18.
 4. The method of claim 1 in which removal of those cells thatexpress at least one human MHC class Ia antigen is carried out with anantibody against RT1A.